Aurora kinase-A overexpression in mouse mammary epithelium induces mammary adenocarcinomas harboring genetic alterations shared with human breast cancer.

Recent data from The Cancer Genome Atlas analysis have revealed that Aurora kinase A (AURKA) amplification and overexpression characterize a distinct subset of human tumors across multiple cancer types. Although elevated expression of AURKA has been shown to induce oncogenic phenotypes in cells in vitro, findings from transgenic mouse models of Aurora-A overexpression in mammary glands have been distinct depending on the models generated. In the present study, we report that prolonged overexpression of AURKA transgene in mammary epithelium driven by ovine ß-lactoglobulin promoter, activated through multiple pregnancy and lactation cycles, results in the development of mammary adenocarcinomas with alterations in cancer-relevant genes and epithelial-to-mesenchymal transition. The tumor incidence was 38.9% (7/18) in Aurora-A transgenic mice at 16 months of age following 4-5 pregnancy cycles. Aurora-A overexpression in the tumor tissues accompanied activation of Akt, elevation of Cyclin D1, Tpx2 and Plk1 along with downregulation of ERα and p53 proteins, albeit at varying levels. Microarray comparative genomic hybridization (CGH) analyses of transgenic mouse mammary adenocarcinomas revealed copy gain of Glp1r and losses of Ercc5, Pten and Tcf7l2 loci. Review of human breast tumor transcriptomic data sets showed association of these genes at varying levels with Aurora-A gain of function alterations. Whole exome sequencing of the mouse tumors also identified gene mutations detected in Aurora-A overexpressing human breast cancers. Our findings demonstrate that prolonged overexpression of Aurora-A can be a driver somatic genetic event in mammary adenocarcinomas associated with deregulated tumor-relevant pathways in the Aurora-A subset of human breast cancer.

SureSelectXT RNA Direct: A Technique for Expression Analysis Through Sequencing of Target-Enriched FFPE Total RNA.

Gene expression profiling of samples from biobanks requires a method that can be used with intact as well as partially degraded RNA. High throughput applications can benefit from reducing the number of processing steps including eliminating the poly(A) selection and ribosomal depletion steps. When performing targeted capture, we have found that we can eliminate the upfront poly(A) selection/ribosomal depletion steps that cause bias in standard mRNA-Seq workflows. This target enrichment solution allows for whole transcriptome or customized content to characterize differential gene expression patterns (especially for mid/low level transcripts). Protocol modifications to the Agilent Strand-Specific RNA Library Prep kit resulted in a new workflow called "RNA Direct" that generates RNA-Seq data with minimal ribosomal contamination and good sequencing coverage. Using RNA isolated from a set of matched samples including fresh frozen (FF) or formalin-fixed, paraffin-embedded (FFPE) from tumor/normal tissues we generated high-quality data using a protocol that does not require upfront ribosomal depletion or poly(A) selection. Using SureSelectXT RNA Direct protocol (RNA Direct) workflow, we found transcripts to be upregulated or downregulated to similar degrees with similar confidence levels in both the FF and FFPE samples, demonstrating the utility for meaningful gene expression studies with biobank samples of variable quality.